Method of removing protein from a water soluble gum and encapsulating cells with the gum

ABSTRACT

Contaminating protein is removed from a water soluble gum such as alginate by dialyzing a solution of the gum against a solution of a disulphide bond reducing agent. Purifying the gum by removing antigenic protein improves biocompatability of the gum for making biocompatible capsules containing cells such as mammalian cells. The reducing agent is preferably dithiothreitol, dithioerythritol or 2-mercaptoethanol. Dialyzing is preferably carried out for more than 1 hour and more preferably twice, each time for 2 hours. Cells are encapsulated by forming a suspension of cells in an aqueous solution of the gum, forming droplets from the suspension, gelling the droplets with a multivalent cation, contacting the gelled droplets with a polymer containing cationic groups, such as poly-1-lysine chloride, that cross-link with anionic groups of the gum to form a semi-permeable membrane around the droplets, and coating the membrane with a layer of the dialyzed gum. Islets of Langerhans cells can be encapsulated for implanting to produce insulin. In addition to encapsulating cells, the purified gum can be used in making dressings, swabs, sutures, stitches, controlled or sustained release devices and artificial vascular grafts that come in contact with body fluids such as blood.

This invention concerns a method of making biocompatible capsulescontaining cells for implantation into a body which lacks the normalfunctioning of those cells.

A common problem caused by the implantation of foreign cells and tissueis that of immunological rejection by the recipient. Attempts toovercome this problem commonly involve treating a patient withimmunosuppressive drugs. However, this is known to be dangerous as thesetypes of drugs have many side effects. Alternatively, the tissue may betreated in some way so as not to provoke an immune response.

With regard to the latter, means of encapsulating cells, particularlyislet cells, have been developed as a method of immunoprotection, usingrelatively nonantigenic material (Lim and Sum, 1980, Science, 210,908-910; Lim, U.S. Pat. No. 4,409,331). In their method, a suspension ofislet cells in sodium alginate solution was formed into droplets andcoated with poly-1-lysine to form a membrane permeable to smallmolecules such as glucose and insulin, but impermeable to largemolecules such as immunoglobulins and the cells of the immune system.

The method was modified by O'Shea et al (1984, Biochimica et BiophysicaActa, 804, 133-136) by coating the capsules with an additional outerlayer of sodium alginate in order to further minimise the inflammatoryresponse.

Although some transplantation of encapsulated cells has been moderatelysuccessful showing normoglycaemia in diabetic mice two weeks aftertransplantation of encapsulated islet cells (Calafiore et al, DiabetesResearch and Clinical Practice, 1988, 5 suppl 1, S334; Tze and Tai,1982, Transplantation, 33, 563-564; Ricker and Stockberger, 1986,Diabetes, 35, Suppl 1, 61A), the capsules, on removal, showedinfiltration of monocytes and macrophages, ie. severe inflammatoryreaction.

A recent study (Clayton et al, Diabetes Research 1990 14 127-132)testing the immune response of rats to implanted empty capsules showedthat the composition of the outer layer of the capsule affects theseverity of the response, with M-alginate (high mannuronic acid sodiumalginate), provoking the least immune response. However, even thisreduced response resulted in macrophage and fibroblast infiltrationafter only 3 weeks.

The present invention overcomes, or at least relieves, the problemsaforesaid.

According to the present invention there is provided a method of makingbiocompatible capsules containing cells comprising the steps:

i) suspending the cells in an aqueous medium containing a pre-dialysedwater soluble gum which has plural anionic moieties but is substantiallyprotein free;

ii) forming the suspension into droplets containing the cells;

iii ) subjecting the droplets to a solution of multivalentphysiologically compatible cations to gel the droplets, encapsulatingsaid cells;

iv) subjecting said gelled droplets to a polymer containing cationicgroups which cross link with said anionic groups to form asemi-permeable membrane;

v) coating said semi-permeable membrane with a layer of saidpre-dialysed water soluble gum.

The water soluble gum may be sodium or other water soluble alginate,such as potassium alginate.

The alginate may be predialysed with phosphate buffered saline (PBS)containing dithiothreitol (DTT), which helps break down any associatedprotein by breaking the disulphide bonds present therein. The alginatemay be predialysed with PBS containing any chemical capable of reducingdisulphide bonds in associated protein, for example, dithioerythritol,2-mercaptoethanol, and the like.

"Substantially protein free" in this context does not necessarily meanthat protein associated with the alginate or other gum is totallyremoved. Unexpectedly, it is found that it is sufficient if disulphidebonds in the associated protein are broken.

The dithiothreitol concentration may be greater than 0.1 mg/ml,preferably 0.6 mg/ml, other chemicals in corresponding amounts.

The alginate may be dialysed for more than 1 hour, preferably twice for2 hours against PBS containing DTT, or any chemical capable of reducingdisulphide bonds, and against PBS alone for more than 65 hours, toensure efficient removal of contaminating antigenic protein.

The cells may be suspended in normal saline containing the purifiedalginate.

The cell suspension may contain more than 1% w/v of the pre-dialysed,purified alginate, preferably 1.5% w/v.

The cells to be encapsulated may be mammalian cells such as insulinsecreting islet of Langerhans cells, useful for implantation intodiabetic patients.

Where islets of Langerhans cells are to be used for transplantation, 1to 2 whole islets may be encapsulated.

The suspension of cells and alginate may be formed into droplets between0.3 and 1.0 mm in diameter by dropping from a syringe into a solutioncontaining cations which gels the droplets, encapsulating said cells.

The solution containing cations may be calcium chloride at aconcentration greater than 0.5% w/v, preferably 1.1% w/v.

The polymer containing cationic groups to which the gelled droplets aresubjected to may be poly-1-lysine chloride.

The molecular weight of the poly-1-lysine chloride may be greater than3000, preferably 20,000

The poly-1-lysine chloride may be dissolved in normal saline solution ata concentration of more than 0.01% w/v, preferably at a concentration of0.05% w/v.

The gelled droplets may be subjected to the poly-1-lysine chloridesolution for more than 1 minute to form a semi-permeable membrane,preferably for 6 minutes.

The semi-permeable membrane thus formed may be coated with a layer ofpre-dialysed water soluble gum by subjecting the semi-permeable membraneto a solution containing more than 0.1% w/v of the gum for more than 1minute.

The solution may contain 0.15% w/v pre-dialysed sodium alginate innormal saline.

The semi-permeable membrane may be subjected to the solution for 4minutes.

The invention also comprises a biocompatible capsule containing cellsmade by the method described herein.

The invention also comprises a method for purifying water soluble gumssuch as water soluble alginates such as sodium alginate and potassiumalginate comprising predialysing the gum with a solution containing adisulphide bond reducing agent, and also comprises a gum purified bysuch method.

By purification in this context is meant eliminating or substantiallyreducing bio-activity due to associated proteinaceous material to renderthe gum (assumed per se biocompatible) sufficiently biocompatible forthe contemplated end use.

In addition to its utility for encapsulating islets of Langerhans cellsfor implantation into diabetic patients, alginate (or otherbiocompatible water soluble gum) purified by the method of the inventionis useful in any situation where an alginate is in direct contact withbody fluids, including blood.

Where dressings, swabs, sutures and stitches and the like are introducedinto the body during surgery and left there post-operation, alginatepurified by the method of the invention is particularly useful.Dressings may be woven or knitted fabrics constructed from alginatefibres. They might also be a non-woven fabric pad made from teasedalginate fibres, such as cotton wool, or a moist sheet or formed slab orshape made from an alginate gel or film. Swabs may be non-wovenassemblies of alginate fibres. Sutures and stitches may be made fromsingle or multiple filament spun threads of alginate.

Similar dressings that are used to cover open surface wounds may also beprepared using alginate purified by the method of the invention.Alginate dressings are used as hemostats and for moist wound management.Alginate powder is also used as a hemostat for open wounds (e.g. topromote fast clotting for sports use).

Dressings and patches that are implanted below the skin and whichcontain active ingredients may also be prepared using alginate purifiedby the method of the invention.

Controlled or sustained release devices which are implanted into thebody or injected rather than going through the normal digestive systemmay also be prepared using alginate purified by the method of theinvention.

Alginate-containing capsules or other devices, including devicesimplanted into the body, whereby the alginate provides a membrane tosieve by molecular size and control the flow of chemicals into and outof the capsule, may be prepared using alginate purified by the method ofthe invention.

These alginates may also be used for coating artificial vascular grafts(e.g. Dacron (RTM) grafts) which are implanted into a patient.

The invention will be further apparent from the following description,with reference to the experiment detailed herein, which shows, by way ofexample only, one form of the method of making biocompatible capsulescontaining cells embodying the invention.

Protocol for making biocompatible capsules containing islet ofLangerhans cells

1. Purification of sodium alginate.

Sodium alginate (supplied by Kelco International) was made up to 1.5%(w/v) solution in normal saline (0.9% w/v sodium chloride). 10 ml ofthis solution was put into a dialysis sac (Sigma, Cat. No. 250-11),which retains proteins with a molecular weight greater than 12,000. Thesodium alginate was dialysed against phosphate buffered saline (PBS; 140mM NaCl, 35 mM K₂ PO₄, pH 7.3) containing dithiothreitol (DTT) for 2hours and dialysed for a further 2 hours against fresh PBS+DTT, afterwhich it was dialysed against PBS alone for a total of approximately 70hours, with fresh PBS at 1 and 65 hours.

The sodium alginate was analysed for the presence of contaminatingantigenic protein by polyacrylamide gel electrophoresis. This analysisconfirmed that the sodium alginate was indeed protein free. Protein wasnot removed from sodium alginate when it was dialysed against PBS withno DTT in an identical procedure to that stated above. This suggeststhat the DTT is essential for the efficient removal of protein fromscdium alginate.

2. Protocol for encapsulation

Islets were removed from either rats or humans and suspended in a 1.5%w/v solution of purified (ie. protein free) sodium alginate in normalsaline. The solution was mixed and drawn up into a 2 ml syringe via aquill and attached to an infusion pump. The infusion pump was set to thedesired rate, normally 1 ml/min, and the drops of scdium alginate/isletsuspension dropped into a 1.1% w/v solution of calcium chloride from aheight of 7 cm so that the drops would be spherical on entering thecalcium chloride solution. On entering the solution the sodium alginategels to encapsulate the islets, usually containing 1-2 islets percapsule, the capsules being in the range 0.3-1.0 mm in diameter. Thegelled capsules are washed with 25 ml volumes of 0.55% CaCl₂ ; 0.27%CaCl₂ ; 0.85% NaCl. The capsules are then placed in 0.1% CHES (pH 8.2 inNaCl) for 3 minutes before being suspended and agitated in 25 ml of0.05% poly-1-lysine hydrochloride (MW 20,000) in NaCl for 6 minutes. Thecationic groups on the poly-1-lysine crosslink with the anionic groupson the sodium alginate capsule to form a semi-permeable membrane. Themolecular weight of the poly-1-lysine is selected to form asemi-permeable membrane of the desired permeability, in this casesufficient to allow out insulin but not to allow in immunoglobulins andother cells of the immune system.

The capsules are then washed with 25 ml of 0.1% CHES and a further 25 mlof 0.85% NaCl before an outer coat of purified sodium alginate is addedto the capsules in order to further increase their biocompatability.This is done by suspending the washed capsules in a 0.15% w/v sodiumalginate solution (in normal NaCl) for 4 minutes with agitation. Thecoated capsules are then washed with 25 ml 0.9% NaCl, suspended in 10 mlof 55 mM sodium citrate for 6 minutes, washed again with 25 ml 0.9%NaCl, washed twice with cell culture medium before being suspended incell culture medium prior to implantation.

Initial results using implanted encapsulated islets in rats confirmedthe greatly increased biocompatability of the capsules using "purified"sodium alginate. This gives great hope for the future implantation ofhuman islets into diabetic patients, which would mean that they couldproduce their own insulin, eliminating the need for daily injections.

The enhanced glucose control provided by transplanted islets compared toinjected insulin may potentially avoid the complications such asblindness and kidney failure which affect a proportion ofinsulin-dependent diabetic patients.

We claim:
 1. A method for substantially removing protein from watersoluble gums containing protein comprising dialyzing a solution ofwater-soluble gum containing protein against a solution containing adisulphide bond reducing agent for a period of time sufficient tosubstantially remove protein from the gum.
 2. A method according toclaim 1, in which the disulphide bond reducing agent is contained in aphosphate buffered saline.
 3. A method according to claim 1, in whichthe reducing agent comprises dithiothreitol.
 4. A method according toclaim 1, in which the reducing agent comprises dithioerythritol.
 5. Amethod according to claim 1, in which the reducing agent comprises2-mercaptoethanol.
 6. A method according to claim 3, in which thedithiothreitol concentration is greater than 0.1 mg/ml.
 7. A methodaccording to claim 6, in which the dithiothreitol concentration is 0.6mg/ml.
 8. A method according to claim 1, in which said dialyzing iscarried out at least once for more than one hour.
 9. A method accordingto claim 8, in which said dialyzing is carried out twice, each time for2 hours.
 10. A method according to claim 1, followed by an analysisconfirming that the water-soluble gum is substantially protein free. 11.A method according to claim 10, in which the analysis is carried out bypolyacrylamide gel electrophoresis.
 12. A method of making biocompatiblecapsules containing cells comprising the steps:i) dialyzing a solutionof a water soluble gum which has plural anionic groups and containsprotein against a solution of a disulphide bond reducing agent for aperiod of time sufficient to make the water soluble gum substantiallyprotein free, and suspending cells in an aqueous medium containing thedialyzed water soluble gum to produce a cell suspension; ii) forming thesuspension into droplets containing the cells; iii) subjecting thedroplets to a solution of multivalent physiologically compatible cationsto gel the droplets, encapsulating said cells; iv) subjecting saidgelled droplets to a solution of a polymer containing cationic groupswhich cross link with said anionic groups to form a semi-permeablemembrane; and v) coating said semi-permeable membrane with a layer ofsaid dialyzed water soluble gum.
 13. A method according to claim 12,wherein the water soluble gum is a water-soluble alginate.
 14. A methodaccording to claim 12, wherein the reducing agent comprisesdithiothreitol.
 15. A method according to claim 12, wherein the reducingagent comprises dithioerythritol.
 16. A method according to claim 12,wherein the reducing agent comprises 2-mercaptoethanol.
 17. A methodaccording to claim 14, wherein the dithiothreitol concentration isgreater than 0.1 mg/ml.
 18. A method according to claim 17, wherein thedithiothreitol concentration is 0.6 mg/ml.
 19. A method according toclaim 13, wherein said dialyzing is carried out at least once for morethan 1 hour.
 20. A method according to claim 19, wherein said dialyzingis carried out twice, each time for 2 hours.
 21. A method according toclaim 12, wherein the aqueous medium is normal saline.
 22. A methodaccording to claim 13, wherein the cell suspension contains more than 1%w/v of said water-soluble alginate.
 23. A method according to claim 22,wherein the cell suspension contains 1.5% w/v of said water-solublealginate.
 24. A method according to claim 12, wherein the cells aremammalian cells.
 25. A method according to claim 24, wherein the cellsare islets of Langerhans cells.
 26. A method according to claim 25,wherein 1 to 2 islets are encapsulated.
 27. A method according to claim12, wherein the suspension is formed into droplets with a syringe andthe gelled droplets are between 0.3 and 1.0 mm in diameter.
 28. A methodaccording to claim 27, wherein the solution containing cations iscalcium chloride at a concentration greater than 0.5% w/v.
 29. A methodaccording to claim 28, wherein the calcium chloride solution is 1.1%w/v.
 30. A method according to claim 12, wherein the polymer containingcationic groups is poly-1-lysine chloride.
 31. A method according toclaim 30, wherein the molecular weight of the poly-1-lysine chloride isgreater than
 3000. 32. A method according to claim 31, wherein themolecular weight of the poly-1-lysine chloride is 20,000.
 33. A methodaccording to claim 30, wherein the poly-1-lysine chloride is in normalsaline solution at a concentration of more than 0.01% w/v.
 34. A methodaccording to claim 33, wherein the concentration of poly-1-lysinechloride is 0.05% w/v.
 35. A method according to claim 12, wherein thepolymer is poly-1-lysine chloride and the gelled droplets are subjectedto the poly-1-lysine chloride solution for more than 1 minute.
 36. Amethod according to claim 35, wherein the gelled droplets are subjectedto the poly-1-lysine chloride solution for 6 minutes.
 37. A methodaccording to claim 12, wherein the semi-permeable membrane is coatedwith a layer of said dialyzed water soluble gum by subjecting thesemi-permeable membrane to a solution containing more than 0.1% w/v ofsaid dialyzed water-soluble gum for more than 1 minute.
 38. A methodaccording to claim 37, wherein the solution contains 0.15% wt/v of saiddialyzed water-soluble gum.
 39. A method according to claim 37, whereinthe semi-permeable membrane is subjected to the solution of saiddialyzed water-soluble gum for 4 minutes.
 40. A biocompatible capsulecontaining cells made by the method described in claim 12.